Comparative proteomic analysis reveals that T3SS, Tfp, and xanthan gum are key factors in initial stages of Citrus sinensis infection by Xanthomonas citri subsp. citri

Abstract

The bacteria Xanthomonas citri subsp. citri (Xac) is the causal agent of citrus canker. The disease symptoms are characterized by localized host cell hyperplasia followed by tissue necrosis at the infected area. An arsenal of bacterial pathogenicity- and virulence-related proteins is expressed to ensure a successful infection process. At the post-genomic stage of Xac, we used a proteomic approach to analyze the proteins that are displayed differentially over time when the pathogen attacks the host plant. Protein extracts were prepared from infectious Xac grown in inducing medium (XAM1) for 24 h or from host citrus plants for 3 or 5 days after infection, detached times to evaluate the adaptation and virulence of the pathogen. The protein extracts were proteolyzed, and the peptides derived from tryptic digestion were investigated using liquid chromatography and tandem mass spectrometry. Changes in the protein expression profile were compared with the Xac genome and the proteome recently described under non-infectious conditions. An analysis of the proteome of Xac under infectious conditions revealed proteins directly involved in virulence such as the type III secretion system (T3SS) and effector proteins (T3SS-e), the type IV pilus (Tfp), and xanthan gum biosynthesis. Moreover, four new mutants related to proteins detected in the proteome and with different functions exhibited reduced virulence relative to the wild-type proteins. The results of the proteome analysis of infectious Xac define the processes of adaptation to the host and demonstrate the induction of the virulence factors of Xac involved in plant-pathogen interactions.

Comparative proteomic analysis of Xanthomonas citri ssp. citri periplasmic proteins reveals changes in cellular envelope metabolism during in vitro pathogenicity induction

“…b), proteomic analysis was performed in cells at the late logarithmic phase because a greater number of cells can be recovered at this stage, increasing the feasibility of subproteomic analysis. This time point also corresponds to the third day of infection in plants, when the recovered bacterial cell mass is sufficient for further analysis (Facincani et al ., ). At the end of the logarithmic phase, the hrp genes tested ( hrpD6 , hrpG , hrpB7 , hpa1 and hrpX ) were still more highly expressed than in the control (Fig.…”

“…The global proteome of XAC cells grown in nutrient broth (NB) medium, a rich and pathogenicity‐non‐inducing medium, has been analysed previously using two‐dimensional gel electrophoresis followed by liquid chromatography coupled with tandem mass spectrometry (2‐DE LC‐MS/MS), providing the first map of XAC expressed proteins (Soares et al ., ). In addition, the XAC total proteome has been analysed under in vitro and in vivo infectious conditions using multidimensional protein identification technology (MudPIT), confirming the involvement of many proteins related to type III and IV secretion systems (T3SS and T4SS) and xanthan gum biosynthesis in XAC pathogenicity (Facincani et al ., ).…”

“…The composition of XAM‐M is based on the well‐known pathogenicity‐inducing media for Xanthomonas spp. : XVM2 (Wengelnik et al ., ) and XAM‐1 (Facincani et al ., ). We demonstrated that XAM‐M is able to induce hrp ( h ypersensitive r esponse and p athogenicity) genes in XAC.…”

Positive selection is the main driving force for evolution of citrus canker-causing Xanthomonas

“…For example, gumC and gumB were found to be under positive selection. The gum genes, responsible for biosynthesis of xanthan gum, are crucial for Xac to form biofilms and survive on the surface of citrus tissues to initiate infection (Facincani et al, 2014).…”

Identification of New Genes Related to Virulence of <i>Xanthomonas axonopodis</i> Pv. <i>Citri</i> during Citrus Host Interactions

“…With the aim of gaining a better understanding of the repertoire of genes and proteins associated with Xac adaptation and virulence-induction in citrus, a series of studies have described the effect of mutations in specific genes impairing pathogen virulence and physiological responses of plant host under pathogen infection [9] [10] [11] [12] [13]. However, there are few published studies that jointly describe the discovery of a new gene repertoire related to pathogenicity and virulence in the Xac genome, which represents a breakthrough in the understanding of the relationship of this phytopathogen with its compatible hosts.…”